CN106635999A - Establishment and application of MMHRL1 transgenic mouse liver tumor cell line - Google Patents

Abstract

The invention provides the establishment and application of H- ras 12V transgenic male mouse liver tumor cell line MMHRL1. Specifically, the H- ras 12V transgenic male mouse liver tumor cell line MMHRL1 provided by the present invention has stable properties, can be stably passed down for multiple times, and is suitable for establishing liver tumor animal models, which is the basic and preclinical application research of liver cancer ideal cell line. In addition, the H- ras 12V transgenic male mouse liver tumor cell line MMHRL1 expresses the H- ras mutant oncogene and has highly activated Ras/MAPK and PI3K/AKT/mTOR signaling pathways. The invention also provides the application of the cell line in establishing model animals, screening drugs and the like.

Description

Establishment and application of MMHRL1 transgenic mouse liver tumor cell line

technical field

The present invention relates to the fields of biology and oncology, and more specifically relates to a novel H-ras12V transgenic male mouse liver tumor cell line MMHRL1, its establishment method and application.

Background technique

The occurrence and development of human diseases is a very complex process. In-depth study of the pathogenesis of various diseases and the effect of clinical drug treatment in order to achieve the purpose of preventing and curing various diseases is the unremitting goal of human beings. However, based on humanitarianism, direct experiments on patients cannot be used. In accordance with the Animal Protection Law, animal experiments can be used to conduct in-depth research on the occurrence and development of various diseases and life phenomena, so as to achieve the purpose of controlling the occurrence and development of human diseases, relieving patients' pain, and finally curing diseases.

In the long-term exploration and practice of human beings, it has been found that it is difficult to promote the rapid and in-depth development of medicine in the study of clinical diseases by taking healthy and sick human bodies as the research objects, and there are many problems in humanities, ethics, and morality. In addition, clinical disease research still has limitations such as the accumulation of a large number of samples and long-term observation.

Animal model of human diseases refers to experimental animals and materials with human disease performance established in biomedical research. It is a crucial experimental method and means in modern biomedical research to study the occurrence and development of human diseases and the treatment measures through animal models of human diseases. Among them, cell culture has a history of more than 100 years and is an important method for in vitro basic medical research.

With the change of people's life style, the process of social industrialization and the prolongation of life expectancy, the incidence of tumor has also increased, which has become one of the main reasons threatening human life and health. Liver cancer, known as the "king of cancers", is a key disease in my country to prevent and treat. However, its causes are numerous, its process is complex, and its cure rate is poor. The mechanism is still unclear and needs to be further elucidated.

At present, dozens of human liver cancer cell lines have been established. However, due to the complex source background and different molecular mechanisms of each liver cancer cell line, it is not conducive to accurate analysis of the etiology. In addition, these cell lines also have the disadvantages of limited tumorigenic ability and unstable passage, which are not conducive to the basic research of liver tumors. Mice are typical animal models of human diseases, and the etiology of liver cancer can be deeply analyzed by establishing animal models of primary liver tumors induced by a single cause. However, due to the high cost, time-consuming and labor-intensive experiments in mice, and the complex influencing factors, it is urgent to establish corresponding mouse liver tumor cell lines. But in contrast, the establishment of mouse liver tumor cell lines is more difficult. Therefore, there are currently only a handful of mouse liver tumor cell lines available.

Therefore, there is an urgent need in this research field to develop mouse liver tumor cell lines with clear etiology, stable passage, high tumorigenicity, and suitable for establishing various animal models.

Contents of the invention

The purpose of the present invention is to provide a liver tumor cell line for basic research and clinical treatment of liver tumors. The cell line has stable transmission, good tumorigenicity and is suitable for establishing various animal models of liver tumors.

Another object of the present invention is to provide the preparation method and application of the H-ras12V transgenic male mouse liver tumor cell line.

In the first aspect of the present invention, a H-ras12V transgenic male mouse liver tumor cell is provided, the liver tumor cell is H-ras12V transgenic male mouse liver tumor cell MMHRL1, which is preserved in China Type Culture Collection Center, the deposit number is CCTCC NO: C2016192.

In the second aspect of the present invention, progeny cells of the H-ras12V transgenic male mouse liver tumor cells described in the first aspect of the present invention are provided, and the progeny cells described can cause the formation of liver tumors in nude mice.

In another preferred example, the described progeny cells substantially or completely retain the characteristics of the parental H-ras12V transgenic male mouse liver tumor cell MMHRL1.

In another preferred example, the H-ras12V transgenic male mouse liver tumor cell MMHRL1 or its progeny cells have the following characteristics:

(a) 100% cells express H-ras12V transgene;

(b) It has the ability to form subcutaneous tumors in immunodeficient mice.

In the third aspect of the present invention, the use of the above-mentioned H-ras12V transgenic male mouse liver tumor cell MMHRL1 or its progeny cells of the present invention is provided, and they can be applied to generate liver tumors in nude mice.

In a fourth aspect of the present invention, a method for establishing a liver tumor animal model is provided, comprising the steps of:

(a) inoculating 1×10 ⁶ -1×10 ⁸ of the H-ras12V transgenic male mouse liver tumor cell MMHRL1 or its progeny cells of the present invention into nude mice;

(b) Feeding the nude mice in step (a) for 14-90 days to obtain a liver tumor animal model.

In another preferred example, the inoculation is at the following sites: liver, abdominal cavity, tail vein, subcutaneous site or a combination thereof.

In another preferred example, nude mice are cultured for 7-100 days in step (b) to obtain a liver tumor animal model.

In the fifth aspect of the present invention, there is provided a method for culturing liver tumor cells in vitro, comprising the steps of: cultivating the above-mentioned H-ras12V transgenic male mouse liver tumor cell MMHRL1 or its progeny of the present invention in a suitable medium cell.

In a sixth aspect of the present invention, a method for screening candidate drugs for treating liver tumors is provided, comprising the steps of:

(a) inoculating 1×10 ⁶ -1×10 ⁸ of the H-ras12V transgenic male mouse liver tumor cell MMHRL1 or its progeny cells of the present invention into nude mice;

(b) raising the nude mice in step (a) for 14-90 days to obtain a liver tumor animal model;

(c) Applying the test drug to the animal model of liver tumor obtained in step (b), and comparing it with the animal model of liver tumor obtained in step (b) without applying the drug, wherein the application leads to improvement of liver tumor symptoms or cure The drug being tested is a drug candidate for treating liver tumors.

In another preferred example, in step (c), the administration method of the test compound can be selected from the following: local administration to the lesion site of the liver tumor, intravenous administration, or oral administration.

In the seventh aspect of the present invention, a liver tumor model animal prepared by the above-mentioned method of the present invention is provided.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions, limited to Space, not described here one by one.

Description of drawings

Figure 1 shows the culture of the H-ras12V transgenic male mouse cell line MMHRL1 of the present invention. Figure 1A shows the situation of cells crawling out of the liver tumor tissue mass to form tumor cell clusters when the semi-dry method was cultured for 12 days (200×); Figure 1B shows the H-ras12V transgenic male mouse cell line MMHRL1 cells of the present invention were cultured to the 20th generation The growth morphology of (200×).

Figure 2 shows the cell morphology and proliferation of the H-ras12V transgenic male mouse cell line MMHRL1 of the present invention. Figure 2A shows the cell morphology (400×) of the H-ras12V transgenic male mouse cell line MMHRL1 of the present invention; Figure 2B shows the proliferation of the H-ras12V transgenic male mouse cell line MMHRL1 of the present invention.

Figure 3 shows the genotype identification and expression level detection of the H-ras12V transgene of the H-ras12V transgenic male mouse cell line MMHRL1 of the present invention.

Figure 4 shows the Western Blot detection results of the signaling pathway activation level of the H-ras12V transgenic male mouse cell line MMHRL1 of the present invention.

Figure 5 shows the experimental results of tumor formation in nude mice subcutaneously inoculated with the H-ras12V transgenic male mouse cell line MMHRL1 of the present invention.

detailed description

After extensive and in-depth research, the present inventors primary cultured a liver tumor sample of an H-ras12V transgenic male mouse, and finally established the H-ras12V transgenic male mouse cell line MMHRL1 with unlimited proliferation ability. Completed this invention on this basis.

Specifically, the inventors cultured the liver tumor tissue of a H-ras12V transgenic male mouse in vitro to establish corresponding in vitro cell lines.

Carry out relevant biological detection and identification on cell lines. A purebred H-ras12V transgenic male mouse cell line MMHRL1 was obtained through screening. The cell line grows stably (it has been stably passaged to 80 passages), and the morphological characteristics of the cell line under the light microscope are uniform.

Animal experiments have shown that the H-ras12V transgenic male mouse cell line MMHRL1 can cause nude mice to develop tumors after inoculation, and its histopathological morphology is consistent with H-ras12V transgenic male mouse liver tumor tissue.

The H-ras12V transgenic male mouse cell line MMHRL1 of the present invention can not only prepare liver tumor animal models, but also can be used to screen candidate drugs for treating liver tumors.

A preferred screening method for candidate drugs for the treatment of liver tumors (or liver tumor cell growth), comprising the steps of:

(a) In the test group, add a test compound to the culture system of the H-ras12V transgenic male mouse liver tumor cell MMHRL1, and observe the cell number and/or growth of the H-ras12V transgenic male mouse liver tumor cell MMHRL1; In the control group, no test compound was added to the culture system of H-ras12V transgenic male mouse liver tumor cells MMHRL1, and the cell number and/or growth of H-ras12V transgenic male mouse liver tumor cells MMHRL1 were observed;

Wherein, if the number or growth rate of H-ras12V transgenic male mouse cells MMHRL1 in the test group exceeds that of the control group (there is a significant difference), it is suggested that the test candidate drug has a promoting effect on the growth or proliferation of liver tumor cells.

In another preferred example, the described less than means that the ratio of the number of the test group to the control group or the ratio of the growth rate is ≤0.8, or more preferably the ratio is ≤0.4.

In another preferred example, the described greater than means that the ratio of the number or the growth rate of the test group to the control group is ≥ 2.5, or more preferably the ratio is ≥ 0.5.

In another preferred example, the described significant difference is P<0.05.

A preferred screening method for candidate drugs for the treatment of liver tumors, comprising the steps of:

(a) Liver of 1×10 ⁶ -1×10 ⁸ (preferably 1×10 ⁶ -1×10 ⁷ , more preferably 1×10 ⁷ -1×10 ⁸ ) the above-mentioned H-ras12V transgenic male mouse Tumor cell MMHRL1 was inoculated into nude mice;

(b) raising the nude mice in step (a) for 14-90 days to obtain a liver tumor animal model;

(c) administering the test drug to the animal model of liver tumor in step (b), and comparing it with the animal model of liver tumor in step (b) without administering the test drug, wherein the liver tumor animal model can cause symptoms of liver tumor after administration The test drug that shows improvement or cure can be considered as a candidate drug for the treatment of liver tumors.

The main advantages of the present invention are:

(a) H-ras12V transgenic male mouse liver tumor cell MMHRL1 of the present invention comes from H-ras12V transgenic male mice;

(b) The H-ras12V transgenic male mouse liver tumor cell MMHRL1 of the present invention has stable properties and can be stably passed down multiple times;

(c) The H-ras12V transgenic male mouse liver tumor cell MMHRL1 of the present invention has good tumorigenicity, clear cause of liver tumor pathogenesis and genetic characteristics, and is an ideal cell line for studying liver tumor mechanism and pre-clinical trials;

(d) The H-ras12V transgenic male mouse liver tumor cell MMHRL1 of the present invention can be used for in vitro research on liver tumor-related genes;

(e) The H-ras12V transgenic male mouse liver tumor cell MMHRL1 of the present invention can be used for in vitro screening of liver tumor sensitive drugs.

Below in conjunction with specific embodiment, further elaborate the present invention. It should be understood that the following examples are only used to illustrate the present invention, not to limit the scope of the present invention. In the following examples, for the experimental methods not marked with specific experimental conditions, the experiments were completed according to conventional experimental conditions or according to the experimental conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise noted.

Example 1

Establishment of liver tumor cell MMHRL1 in H-ras12V transgenic male mice

(a) source

The H-ras12V transgenic male mouse liver tumor cell line comes from H-ras12V transgenic male mice, and its specific information is shown in the table below.

Table 1

The primary liver tumor tissue of the H-ras12V transgenic male mouse was obtained, and the liver tumor tissue was directly cultured in vitro, digested and passaged repeatedly, and finally a stable passage H-ras12V transgenic male mouse liver tumor cell line was obtained. Specifically include the following processes:

The above-mentioned H-ras12V transgenic male mice were sacrificed painlessly, and the liver tumors were excised by aseptic technique. Place the liver tumor in a 10 cm culture dish filled with normal temperature sterilized normal saline, fully clean up the tumor capsule, and use a scalpel to cut the liver tumor tissue collected after the above operation into 1× ¹ ×1mm3 tissue pieces , adding liver tumor tissue culture medium to culture by semi-dry method. The composition of liver tumor tissue culture medium is DMEM high glucose medium, supplemented with 10% heat-inactivated fetal bovine serum, 100IU/ml penicillin-streptomycin, and 50mg/ml gentamicin. The medium was changed after 24 hours for the newly inoculated liver tumor tissue pieces, and the medium was changed every 72 hours during the culture of liver tumor cells in H-ras12V transgenic male mice. After 12 days of semi-dry culture, cells can be observed crawling out around the liver tumor tissue block. At this time, conventional cell culture digestion and plating are performed on the above-mentioned liver tumor tissue, and the fibroblasts in the original liver tumor tissue culture are removed during the multiple digestion and plating processes. When the primary cells were first passaged to the 10th passage, a large number of epithelial-like cells with uniform morphology could be obtained, which were named MMHRL1.

At present, H-ras12V transgenic male mouse liver tumor cell MMHRL1 has been passaged to the 80th passage, the cell growth is stable and in good condition, and is still being passaged and cultured (Fig. 1B and Fig. 2A).

The obtained strain of H-ras12V transgenic male mouse liver tumor cell MMHRL1 was deposited with the China Center for Type Culture Collection (CCTCC) (Wuhan, China) on November 08, 2016. The preservation number is CCTCC NO: C2016103, and the preservation address is: Luojia Mountain (No. 299 Bayi Road), Wuchang District, Wuhan City, Hubei Province.

Example 2

Biological characteristics of liver tumor cell MMHRL1 in H-ras12V transgenic male mice

In this example, conventional methods were used to detect the biological characteristics of the purebred H-ras12V transgenic male mouse liver tumor cell MMHRL1 with the preservation number CCTCC NO: C2016103, and the results are as follows:

2.1 Cell Morphology

Observing the liver tumor cells MMHRL1 of H-ras12V transgenic male mice under an ordinary optical microscope, it can be seen that most of the liver tumor cells are round or polygonal, and the cell shape is uniform ( FIG. 2A ). Cell Counting Kit-8 (CCK8 kit) was used to detect the proliferation state of the cell line, and the operation was carried out in full accordance with the kit operation guide ( FIG. 2B ). The cells proliferated stably.

2. Genotype identification of 2H-ras12V transgene

Take the H-ras12V transgenic male mouse liver tumor cell MMHRL1 (preservation number: C2016103) obtained in Example 1, digest it with digestive solution (0.25% trypsin, 0.02% EDTA, pH7.3), collect it, and extract genomic DNA and total RNA for qualitative identification of RAS transgenic genotypes by PCR and RT-PCR detection of RAS gene expression.

(a) RAS transgene PCR qualitative identification method:

Extract H-ras12V transgenic male mouse cell line MMHRL1 genomic DNA, and carry out ordinary PCR. The loading amount is as follows in Table 2, and the results are shown in Figure 3A

Table 2

(b) RAS transgene RT-PCR quantitative detection method:

Extract H-ras12V transgenic male mouse cell line MMHRL1mRNA, synthesize cDNA according to the method in (a), and then perform RT-PCR. The loading amount is as follows in Table 3, and the results are shown in Figure 3B

table 3

2.3 Western Blot detection of H-ras12V transgenic male mouse liver tumor cells MMHRL1 cells

In this example, Western Blot detection was performed on H-ras12V transgenic male mouse liver tumor cell MMHRL1 cells. The result is shown in Figure 4.

The results showed that the MEK/ERK and AKT/mTOR signaling pathways of H-ras12V transgenic male mouse liver tumor cells MMHRL1 cells were significantly activated.

Example 3

Establishment of liver tumor animal model using H-ras12V transgenic male mouse liver tumor cell MMHRL1

Take the H-ras12V transgenic male mouse liver tumor cell MMHRL1 (preservation number: C2016103) obtained in Example 1, collect and count after digesting with digestive solution (0.25% trypsin, 0.02% EDTA, pH7.3), and count according to 1×10 ⁶ or 1×10 ⁸ cells/mouse were inoculated into the subcutaneous site of the right armpit of the mice, and the inoculated mice were 5 nude mice at the age of 5 weeks.

Between 14 and 60 days after inoculation, tumors occurred successively in five mice. The same method as in Example 2 was used for biological detection, and it was confirmed that hepatocellular liver tumors were induced in animal models. The result is shown in Figure 5.

Cell line deposit

The H-ras12V transgenic male mouse liver tumor cell MMHRL1 of the present invention was deposited in the China Center for Type Culture Collection (Wuhan, China) on November 08, 2016, and the preservation number is CCTCC NO: C2016103.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

SEQUENCE LISTING

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Claims (8)

1. a kind of transgenic male mice tumor cell of liver, it is characterised in that the tumor cell of liver is H-ras12V transgenosis Male mice tumor cell of liver MMHRL1, it is deposited in China typical culture collection center, and preserving number is CCTCC NO: C2016103。

2. the daughter cell of H-ras12V transgenic male mices tumor cell of liver as claimed in claim 1, it is characterised in that The daughter cell can result in nude mice and form liver cancer.

3. H-ras12V transgenic male mices tumor cell of liver as claimed in claim 1 or the filial generation described in claim 2 Cell, it is characterised in that the cell has following characteristics：

A () 100% cell expresses H-ras12V transgenosis；

B () has the subcutaneous one-tenth knurl ability of immunodeficient mouse.

4. H-ras12V transgenic male mices tumor cell of liver as claimed in claim 1 or the filial generation described in claim 2 The purposes of cell, it is characterised in that in nude mice.

5. a kind of method for setting up liver tumour animal model, it is characterised in that including step：

A () is by 1 × 10⁶-1×10⁸Claim 1 described in H-ras12V transgenic male mices tumor cell of liver or right It is required that the daughter cell described in 2 is inoculated in nude mice；

B the nude mice of () raising step (a) 14-90 days, obtains liver tumour animal model.

6. a kind of method of in vitro culture H-ras12V transgenic male mice tumor cell of liver, it is characterised in that including step： In suitable culture medium, the H-ras12V transgenic mices tumor cell of liver described in claim 1 or claim 2 institute are cultivated The daughter cell stated.

7. a kind of method that the drug candidate of liver tumour is treated in screening, it is characterised in that including step：

(a) just 1 × 10⁶-1×10⁸Claim 1 described in H-ras12V transgenic male mices tumor cell of liver or power Profit requires that the daughter cell described in 2 is inoculated in nude mice；

B the nude mice of () raising step (a) 14-90 days, obtains liver tumour animal model；

C liver tumour animal model that testing drug is applied to be obtained in step (b) by (), and with (b) the step of non-drug application In the liver tumour animal model that obtains be compared, wherein cause liver tumour symptom to improve after application or the testing drug cured just It is the compound for treating liver tumour.

8. method as claimed in claim 7, it is characterised in that in step (c), by the application method of test compound just like Lower selection：Local delivers medicine to liver tumour lesions position, intravenously administrable, oral administration.